In higher eukaryotic cells, the interaction between ligands (e.g., peptide hormones, growth factors and their analogs) and their receptors is of central importance in the transmission of and response to a variety of extracellular signals. It is generally accepted that peptide hormones and growth factors elicit their biological functions by binding to specific recognition sites (receptors) on the plasma membranes of the target cells. Upon ligand binding, the receptors are believed to undergo a conformational change, triggering intra-cellular responses, which in turn result in the activation or inhibition of some cellular process(es). Ligand analogs fall into two classes: those that mimic the effect(s) of the corresponding natural ligand, termed agonists; and those that block receptor-ligand binding or the effects elicited by the natural ligand, termed antagonists.
Of particular interest is the interaction between platelet-derived growth factor (PDGF) and its receptor(s). PDGF is the major mitogenic protein in serum for mesenchymal cells. It induces cell multiplication or DNA synthesis in cultured smooth muscle cells, fibroblasts and glial cells, is a potent chemoattractant and exhibits other biological activities. The biology of PDGF is reviewed by Ross et al. (Cell 46: 155-169, 1986). PDGF has been shown to play an important role in the wound-healing response (Ross and Glomset, New Eng. J. Med. 295: 369, 1976; Grotendorst et al. J. Clin. Invest. 76: 2323-2329, 1985; Murray et al., U.S. Pat. application Ser. No. 230,190) and is believed to play a causative role in the development of the proliferative lesions of atherosclerosis (Ross and Glomset, ibid.). These activities are mediated by the binding of PDGF to membrane-associated receptors comprising an extracellular binding site, a transmembrane anchor and an intracellular tyrosine kinase domain. Antagonists that block receptors against the action of endogenous PDGF may be useful in the treatment of atherosclerosis or in the inhibition of other conditions involving PDGF-induced aberrant growth patterns. PDGF agonists may be useful for promoting wound healing.
Current methods for screening potential agonists and antagonists involve assaying the binding of radiolabeled compounds to responsive cells, to the membrane fractions of disrupted cells, or to solubilized receptors. Alternatively, compounds may be screened for their ability to compete with a labeled known ligand for cell-surface receptors. For example, Lefkowitz et al. (Biochem. Biophys. Res. Comm. 60: 703-709, 1974), Aurbach et al. (Science 186: 1223-1225, 1974) and Atlas et al. (Proc. Natl. Acad. Sci. USA 71: 4246-4248, 1974) disclose receptor-binding assays for .beta.-adrenergic agonists and antagonists. These assays utilize isolated erythrocyte membranes.
The success of current screening procedures depends in part on the availability of reproducibly high quality preparations of membrane fractions or receptor molecules. The preparation of membrane fractions and soluble receptor molecules often involves extensive manipulations and complex purification steps. Receptors, being integral membrane proteins, require cumbersome purification procedures that include the use of detergents and other solvents that interfere with their biological activity. Furthermore, the large size of typical receptor molecules makes them particularly vulnerable to proteolysis during purification. Production of large amounts of functional receptor proteins by standard techniques of protein chemistry is not economical. The use of membrane preparations in ligand binding assays typically results in low reproducibility due to the variability of such preparations.
In the case of growth factor receptors, ligand-binding assays generally require the isolation of responsive cell lines. Often only a limited population of a responsive cell type is responsive to a particular agent, and such cells may be responsive only under certain conditions. In addition, these cells may be difficult to grow in culture or may possess a low number of receptors.
Most currently available cell types responsive to PDGF contain only a low number of receptors per cell, thus requiring large numbers of cells to assay potential PDGF analogs or antagonists. Such assays are labor-intensive and complex, and do not readily lend themselves to automation and high through-put.
A PDGF receptor that specifically binds the PDGF BB isoform at high affinity (hereinafter referred to as the .beta.-receptor) has been described (Claesson-Walsh et al., Mol. Cell. Biol. 8: 3476-3486, 1988; Gronwald et al., Proc. Natl. Acad. Sci. USA 85: 3435-3439, 1988). Because PDGF can exist in any of three isoforms (AA, AB and BB) or mixtures thereof, this receptor cannot be used to detect all forms of PDGF or analogs thereof.
There is therefore a need in the art for an assay system that permits commercial scale screening of compounds for PDGF agonist and antagonist activity. Such an assay system should be rapid, inexpensive, adaptable to high through-put screening and capable of detecting analogs of all PDGF isoforms. The present invention provides such assay systems, and further provides other related advantages.